PROJECT SUMMARY - PROJECT 3 Long non-coding RNAs (lncRNAs) represent a large and relatively understudied class of RNAs that have great potential to provide novel opportunities to influence gene regulation and cancer biology. The long-term goal of Project 3 is to identify lncRNAs that are over-expressed in breast cancer, define their role in the disease, and develop approaches to manipulate their expression in vivo to impact breast cancer progression. Here, a series of Aims are presented to determine the roles of Malat1 lncRNA, and several recently identified lncRNAs - Mammary Tumor Associated RNAs (MaTARs) - that are expressed in mouse models of luminal B and Her2/neu mammary cancer. We showed that Malat1 is over-expressed in mammary tumors and its genetic loss or knockdown results in differentiation of the primary tumor and a significant reduction in metastasis. Studies are proposed to identify the steps in metastasis that are dependent upon Malat1 and the critical regions of the RNA contributing to its function. Mouse models containing GFP- or bioluminescently-labeled breast tumor cells will be used to follow metastasis and in vivo antisense oligonucleotide (ASO) knockdown will provide a means to reveal the specific dependencies of Malat1 in the metastatic process. 3D tumor organoid cultures will be used to ascertain functional domains within Malat1 and to identify critical regions that are essential for tumorigenesis. The promoters of mammary tumor genes that are impacted upon Malat1 knockdown are enriched in Sox5 and Tcfcp2l1 binding sites. Studies are proposed to examine the role of these transcription factors, in conjunction with Malat1, to regulate the differentiation of primary tumors and the significant reduction in metastasis upon Malat1 knockdown. A series of studies are proposed to determine the function of 4 recently identified MaTARs that are expressed in a mammary tumor-specific manner. Chromatin Isolation by RNA Purification (ChIRP) coupled to deep sequencing or mass spectrometry will be used to identify interacting DNA sequences and proteins. To evaluate the potential of these MaTARs as therapeutic targets genetic knockout mouse models will be established and crossed with mammary tumor models to assess the impact on tumor initiation, progression and metastasis. RNA-seq will be performed on knockout and wild-type tumors to identify global gene expression changes upon MaTAR loss. The expression level and breast cancer sub-type specificity of human MaTAR orthologs has been examined and will be correlated with overall and relapse-free survival, and metastatic incidence, and appropriate candidates will be studied in patient-derived xenograft mouse models. Together, the proposed studies will define the role of several lncRNAs in breast cancer biology, and determine their potential as therapeutic targets to impact breast cancer progression.